Figure 2.

Transient expression of GBD-ROS1_CD and its mutant variants in HEK293 cells. (A-B) Schematic diagrams of effector and reporter constructs used for co-transfection of HEK293 cells. Control effector constructs contain either a mutation in the ROS1 catalytic domain (GBD-ROS1_CDmut) or two mutations in GBD that abolish binding to target UAS sequences (GBDmut-ROS1_CD). Reporter constructs contain the TK promoter fused to the firefly luciferase gene. The targeted version (5xUAS-TK-Luc) includes five copies of GBD binding sites upstream the TK promoter that are absent in the non-targeted control (TK-Luc). (C) Transient expression of GBD-ROS1_CD fusion proteins in HEK293 cells. Western-blot analysis with an anti-Flag antibody was performed in cell extracts (80 μg) prepared 48 h after co-transfection of different effector constructs with either the targeted (upper panels) or the non-targeted (lower panels) methylated reporter plasmid. Actin was used as an input control.